Platelet aggregation inducing substance

ABSTRACT

A platelet aggregation inducing substance containing as an active ingredient a polypeptide having a peptide fragment represented by formula (1) (component A):
 
-(Pro-X-Gly) n -  (1)
 
wherein X represents Pro or Hyp; and n represents an integer of from 20 to 5,000.

TECHNICAL FIELD

The present invention relates to a platelet aggregation inducingsubstance that is capable of being used for evaluation of function ofplatelets.

BACKGROUND ART

Platelets are a biosubstance that is essential for hemostatic actionupon bleeding, and in the case where the function thereof is exhibitedexcessively, thrombus is formed to cause critical diseases, such asmyocardial infarction, cerebral infarction and the like. In the casewhere platelets fail to function, on the contrary, bleeding tendency isinduced. Accordingly, for performing health management and medicaltreatment, it is important to comprehend the state of platelets in bloodby evaluating the function thereof (measurement of aggregation capacityof platelets).

The aggregation capacity of platelets is generally measured in such amanner that aggregation of platelets is induced by adding an aggregationinducing substance, such as collagen and the like, to platelet-richplasma under a low shear rate, and the transmittance increased by theaggregation is measured.

Collagen of animal origin is generally used for measuring aggregationcapacity of platelets, and there are known ones that induce aggregationof platelets in artificially synthesized collagen.

Non-patent Document 1, 2 and 3 disclose a polypeptide -(Gly-Pro-Hyp)₁₀-having a triple helix structure and a substance obtained by crosslinkingthe polypeptide to form a quaternary structure, and also disclose theplatelet aggregation inducing activity of the substances.

The polypeptide -(Gly-Pro-Hyp)₁₀- disclosed in the non-patent documentshas low platelet aggregation inducing activity and cannot be used formeasuring aggregation capacity of platelets.

The substance obtained by crosslinking the polypeptide to form aquaternary structure has higher platelet aggregation inducing activityas compared to the polypeptide and can be used for measuring aggregationcapacity of platelets. For obtaining the substance, however, it isnecessary to perform peptide synthesis reaction including repeateddeprotection and formation of amino acid bond, and it is also necessaryto perform crosslinking. Repetition of the reactions decreases the yieldof the target reaction product. The decrease in yield results inincrease of cost.

[Non-patent Document 1] THE JOURNAL OF BIOLOGICAL CHEMISTRY, May 13,1994, 269(19), 13899-18903

[Non-patent Document 2] The Biochemcial Journal, 1995, 306, 337-344

[Non-patent Document 3] Cardiovascular Research, 1999, 41, 450-457

DISCLOSURE OF THE INVENTION

It is considered that artificially synthesized collagen is advantageousin such points as reproducibility, stability, preservability and thelike, as compared to collagen of animal origin, which has been used as asubstance for inducing aggregation of platelets. However, the syntheticcollagen disclosed in Non-patent Documents 1, 2 and 3 involves suchproblems as low yield, high cost and the like. Furthermore, it involvesfailure in providing sufficient platelet aggregation inducing activitydepending on the structure thereof.

There are cases where aspirin is used for preventing formation ofthrombus, and aspirin in blood impairs the function of the plateletaggregation inducing substance, such as collagen or the like, therebymaking considerably difficult the measurement of platelet aggregationcapacity of a patient taking aspirin. Evaluation of function ofplatelets (measurement of aggregation capacity of platelets) isessential for performing health management and medical treatment asnoted above, and thus such a substance is being demanded thateffectively induces aggregation of platelets even in the presence ofaspirin.

The inventors have made earnest investigations in view of theaforementioned problems associated with the conventional techniques.Consequently, it has been found that a polypeptide represented byformula (1) used in the invention can be obtained by polymerizing atrimer of -(Pro-Hyp-Gly)- in a solvent, thereby minimizing the reactionprocess, which is advantageous in yield and cost, has plateletaggregation inducing activity at such a level that can be sufficientlyused for measurement of aggregation capacity of platelets, and iscapable of effectively inducing aggregation of platelets even in thepresence of aspirin, and thus the invention has been completed.

The invention includes the following aspects (1) to (8).

(1) A platelet aggregation inducing substance containing as an activeingredient a polypeptide having a peptide fragment represented byformula (1) (component A):-(Pro-X-Gly)_(n)-  (1)wherein X represents Pro or Hyp; and n represents an integer of from 20to 5,000.

-   -   (2) The platelet aggregation inducing substance according to the        item (1), wherein the peptide fragment (component A) contains a        peptide unit represented by formula (2) (component B) and a        peptide unit represented by formula (3) (component C), and a        ratio of the component B and the component C (component        B/component C, molar ratio) is in a range of from 95/5 to 0/100:        -(Pro-Pro-Gly)-  (2)        -(Pro-Hyp-Gly)-  (3)    -   (3) The platelet aggregation inducing substance according to the        item (1) or (2), wherein the polypeptide further contains at        least one selected from a peptide unit represented by formula        (4), a peptide unit represented by formula (5), and a peptide        fragment containing at least one of the peptide units (component        D):        —(Y₁—Y₂-Gly)-  (4)        -(Pro-Z₁-Gly-Z₂-Ala-Gly)-(SEQ ID NO:13)  (5)        wherein Y₁ represents Asp or Glu, which may have a carboxyl        group at the γ-position; Y₂ represents Pro or Hyp; Z₁ represents        Gln, Asn, Leu, Ile, Val or Ala; and Z₂ represents Ile or Leu.        The peptide unit represented by formula (5) is selected from the        group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ        ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ        ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.    -   (4) The platelet aggregation inducing substance according to the        item (3), wherein a ratio of the peptide fragment (component A)        and at least one selected from the peptide unit represented by        formula (4), the peptide unit represented by formula (5), and        the peptide fragment containing at least one of the peptide        units (component D) (component A/component D, molar ratio) is in        a range of from 99/1 to 30/70.

(5) The platelet aggregation inducing substance according to one of theitems (1) to (4), wherein the polypeptide exhibits positive Cottoneffect at a wavelength of from 220 to 230 nm and negative Cotton effectat a wavelength of from 195 to 205 nm in a circular dichroic spectrum.

-   -   (6) The platelet aggregation inducing substance according to one        of the items (1) to (5), wherein the polypeptide has a molecular        weight in a range of from 10,000 to 500,000,000.    -   (7) The platelet aggregation inducing substance according to one        of the items (1) to (6), wherein the polypeptide has a particle        diameter in a range of from 0.01 to 1,000 μm.    -   (8) The platelet aggregation inducing substance according to one        of the items (1) to (7), wherein the polypeptide has a viscosity        in a range of from 10 to 10,000 mPa·s, wherein the viscosity is        measured for a 1 wt % aqueous solution of the polypeptide under        a condition of 20° C. with an E-type viscometer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 2.

FIG. 2 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 3.

FIG. 3 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 4.

FIG. 4 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 4.

FIG. 5 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 5.

FIG. 6 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 5.

FIG. 7 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 6.

FIG. 8 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 6.

FIG. 9 The figure shows measurement results of aggregation capacity ofplatelets in Experimental Example 7.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention will be described in detail below.

In the invention, amino acid residues are described by the followingsymbols.

-   Ala: L-alanine residue-   Arg: L-arginine residue-   Asn: L-asparagine residue-   Asp: L-asparaginic acid residue-   Cys: L-cysteine residue-   Gln: L-glutamine residue-   Glu: L-glutamic acid residue-   Gly: glycine residue-   His: L-histidine residue-   Hyp: L-hydroxyproline residue-   Ile: L-isoleucine residue-   Leu: L-leucine residue-   Lys: L-lysine residue-   Met: L-methionine residue-   Phe: L-phenylalanine residue-   Pro: L-proline residue-   Sar: sarcosine residue-   Ser: L-serine residue-   Thr: L-threonine residue-   Trp: L-tryptophan residue-   Tyr: L-tyrosine residue-   Val: L-valine residue

In the specification, the amino acid residues are described with theamino terminal or the N terminal on the left side and the carboxylterminal or the C terminal on the right side according to the ordinaryrule.

The polypeptide as an active ingredient of the invention (which may bereferred to as “active ingredient polypeptide”) is not particularlylimited as far as it is a polypeptide that contains a peptide fragmentrepresented by formula (1) (component A).-(Pro-X-Gly)_(n)-  (1)

The repetition number (n) is an integer of from 20 to 5,000, and ispreferably an integer of from 20 to 3,000 from the standpoint ofplatelet aggregation inducing activity and stability of the structure(triple helix structure).

In formula (1), X represents Pro or Hyp. Accordingly, the peptidefragment (component A) includes a case where it contains only a peptideunit represented by formula (2) (component B), a case where it containsonly a peptide unit represented by formula (3) (component C), and a casewhere it contains the peptide unit (component B) and the peptide unit(component C).-(Pro-Pro-Gly)-  (2)-(Pro-Hyp-Gly)-  (3)

In the invention, the ratio of the component B and the component C(component B/component C, molar ratio) constituting the peptide fragment(component A) is not particularly limited and is preferably in a rangeof from 95/5 to 0/100, more preferably in a range of from 50/50 to0/100, and further preferably in a range of from 10/90 to 0/100, fromthe standpoint of platelet aggregation inducing activity and stabilityof the structure (triple helix structure). In the invention, Hyp is 4Hyp(for example, trans-4-hydroxy-L-proline) residue.

The active ingredient polypeptide may contain at least one peptide unitrepresented by formula (4), may contain at least one peptide unitrepresented by formula (5), or may contain a peptide fragment containingat least one selected from the peptide unit represented by formula (4)and the peptide unit represented by formula (5).—(Y₁—Y₂-Gly)-  (4)-(Pro-Z₁-Gly-Z₂-Ala-Gly)-(SEQ ID NO:13)  (5)

In formula (4), Y₁ may be an amino acid residue having a carboxyl group(for example, an α-amino acid residue having a carboxyl group or thelike), specific examples of which include Asp, Glu and the like, and inthe invention, Y₁ is Asp or Gly, which may have a carboxyl group at theγ-position. Y₂ represents Pro or Hyp.

In formula (5), Z₁ represents Gln, Asn, Leu, Ile, Val or Ala, morepreferably Gln, Asn, Leu, Val or Ala, and further preferably Gln or Leu.Z₂ represents Ile or Leu, and preferably Ile. The peptide unitrepresented by formula (5) is selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11and SEQ ID NO:12.

The combination of Z₁ and Z₂ is not particularly limited, and examplesthereof include a combination where Z₁ is one selected from Gln, Asn,Leu, Ile, Val and Ala (for example, Gln or Leu) and Z₂ is Ile so thatthe peptide unit represented by formula (5) is selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5 and SEQ ID NO:6, and a combination where Z₁ is one selected fromGln, Asn, Leu, Ile, Val and Ala (for example, Gln or Leu) and Z₂ is Leuso that the peptide unit represented by formula (5) is selected from thegroup consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11 and SEQ ID NO:12.

The ratio of the peptide fragment (component A) and at least oneselected from the peptide unit represented by formula (4), the peptideunit represented by formula (5), and the peptide fragment containing atleast one of the peptide units (component D) (component A/component D,molar ratio) is not particularly limited, and is preferably in a rangeof from 99/1 to 30/70, more preferably in a range of from 99/1 to 50/50,and further preferably in a range of from 99/1 to 70/30, from thestandpoint of platelet aggregation inducing activity and stability ofthe structure (triple helix structure).

The active ingredient polypeptide may contain an amino acid residue anda peptide residue other than the above in such a range that does notimpair the physical and biological properties thereof.

The active ingredient polypeptide may be a salt with an inorganic acid(such as hydrochloric acid, sulfuric acid and the like), an organic acid(such as acetic acid, lactic acid, maleic acid, oxalic acid, citric acidand the like), a metal (such as sodium, potassium and the like), or anorganic base (such as trimethylamine, triethylamine and the like). Thesalt compound of the polypeptide may be used in the invention solely oras a combination of two or more of them.

The active ingredient polypeptide preferably exhibits positive Cottoneffect at a wavelength of from 220 to 230 nm and negative Cotton effectat a wavelength of from 195 to 205 nm in a circular dichroic spectrum.The use of the active ingredient polypeptide exhibiting the Cottoneffect provides a platelet aggregation inducing substance havingpractically sufficient activity. The Cotton effect means a phenomenonoccurring due to an optically active substance that exhibits adifference between absorption coefficients to clockwise andanticlockwise circularly polarized light at a particular wavelength.

The active ingredient polypeptide may have a triple helix structure. Thepolypeptide chain forming a triple helix structure may be in a linearform or may have one or more branch. In the case where the polypeptidechain has a branch, the triple helix structure may be formed behind thebranch point, or the branch point may be formed behind the triple helixstructure.

The molecular weight of the active ingredient polypeptide is notparticularly limited, and is preferably in a range of from 10,000 to500,000,000 from the standpoint of platelet aggregation inducingactivity and stability of the structure (triple helix structure).

The particle diameter of the active ingredient polypeptide is notparticularly limited, and is preferably in a range of from 0.01 to 1,000μm, in terms of a diameter measured with a dynamic light scatteringparticle diameter measuring apparatus or a laser diffraction-scatteringparticle diameter measuring apparatus, from the standpoint of plateletaggregation inducing activity.

The viscosity of the active ingredient polypeptide is preferably in arange of from 10 to 10,000 mPa·s, in terms of a viscosity measured for a1 wt % aqueous solution of the polypeptide under a condition of 20° C.with an E-type viscometer, from the standpoint of platelet aggregationinducing activity.

The active ingredient polypeptide may contain only the peptide fragment(component A), and may contain additionally an amino acid residue and analkylene shown below in such a range that does not impair the advantagesof the invention.

Examples of the amino acid residue include at least one amino groupselected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Hyp, Ile,Leu, Lys, Met, Phe, Pro, Sar, Ser, Thr, Trp, Tyr, Val, or a peptideresidue containing several of them bonded to each other.

The alkylene may be linear or branched and is not particularly limited,and examples thereof include an alkylene having from 1 to 18 carbonatoms, and practically an alkylene having from 2 to 12 carbon atoms.

The platelet aggregation inducing substance of the invention may containa substance other than the active ingredient polypeptide in such a rangethat does not impair the advantages of the invention. The substance maybe selected in consideration of preservability, handleability, stabilityof activity and the like and is not particularly limited, and examplesthereof include preservation solvents described later.

A method for producing the active ingredient polypeptide will bedescribed in detail below.

The active ingredient polypeptide may be obtained in any method.However, when a peptide fragment is condensed in a solvent,polymerization proceeds to provide the active ingredient polypeptidewithout repeated deprotection and formation of amino acid bond, andtherefore such a method is preferably employed that a peptide fragmentcontaining amino acids constituting the active ingredient polypeptide issubjected to condensation reaction in a solvent.

The peptide fragment referred in the invention is a peptide fragmentcontaining from 3 to 90 residues. Examples of the peptide fragment thatcan be used for producing the active ingredient polypeptide includepeptide fragments shown below.

No. 1-No. 18

-   wherein o represents an integer of from 1 to 10.    No. 19-No. 67-   wherein p represents an integer of from 1 to 10, and q represents an    integer of from 1 to 10.

Among these, the peptide fragment No. 1 or No. 2 is necessarily used forproducing the active ingredient polypeptide. The peptide fragments Nos.3 to 67 may be used appropriately. Other peptide fragments than theabove may be used in such a range that does not impair the advantages ofthe invention.

The peptide fragment No. 1 or No. 2 and the peptide fragments Nos. 3 to67 or a peptide fragment containing these peptide fragments and otherpeptide fragments than these may be used for producing the activeingredient polypeptide.

The integers o, p and q in the aforementioned peptide fragments and theratios of the peptide fragments used in the condensation reaction arenot particularly limited and are determined based on the ratio of thecomponent B and the component C and the ratio of the component A and thecomponent D of the intended active ingredient polypeptide.

In view of facility of the condensation reaction and availability of thepeptide fragments, o, p and q are preferably each independently aninteger of from 1 to 10, more preferably an integer of from 1 to 5, andparticularly preferably 1.

The peptide fragments Nos. 1 to 67 can be obtained by a known solidphase synthesis method or liquid phase synthesis method.

The condensation reaction of the peptide fragments is generallyperformed in a solvent. The solvent may be one capable of dissolving(partly or wholly dissolving) or suspending the peptide fragments as rawmaterials, and water and an organic solvent can be generally used.Specific examples thereof include water, an amide compound (such asdimethylformamide, dimethylacetamide, hexamethylphosphoramide and thelike), a sulfoxide compound (such as dimethylsulfoxide and the like), anitrogen-containing cyclic compound (such as N-methylpyrrolidone,pyridine and the like), a nitrile compound (such as acetonitrile and thelike, an ether compound (such as dioxane, tetrahydrofuran and the like),an alcohol compound (such as methyl alcohol, ethyl alcohol, propylalcohol and the like), and mixed solvents thereof. Among the solvents,water, dimethylformamide and dimethylsulfoxide are preferably used.

The reaction of the peptide fragments is preferably performed in thepresence of a dehydrating agent (dehydration condensing agent). Uponreacting in the presence of a dehydration condensing agent and acondensation assistant, the condensation reaction smoothly proceedswhile suppressing dimerization and cyclization from occurring.

The dehydration condensing agent is not particularly limited as far asit effectively performs dehydration condensation in the solvent, andexamples thereof include a carbodiimide condensing agent (such asdiisopropylcarbodiimide (DIPC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC=WSCI),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI.HCl),dicyclohexylcarbodiimide (DCC) and the like), a fluorophosphatecondensing agent (such asO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, O-(7-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, benzotriazol-1-yl-oxy-tris-pyrrolidinophosphoniumhexafluorophosphate, benzotriazol-1-yl-tris(dimethylamino)phosphoniumhexafluorophosphide (BOP) and the like), and diphenylphosphorylazide(DPPA).

The dehydration condensing agents may be used solely or as a mixture bycombining two or more of them. Preferred examples of the dehydrationcondensing agent include a carbodiimide condensing agent (such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride).

The condensation assistant is not particularly limited as far as itaccelerates the condensation reaction, and examples thereof include anN-hydroxy polyhydric carboxylic imide compound (such as anN-hydroxydicarboxylic imide compound, e.g., N-hydroxysuccinimide(HONSu), N-hydroxy-5-norbornene-2,3-dicarboxylic imide (HONB) and thelike), an N-hydroxytriazole compound (such as an N-hydroxybenzotriazolecompound, e.g., 1-hydroxybenzotriazole (HOBt) and the like), a triazinecompound, such as 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine(HOObt) and the like, and ethyl 2-hydroxyimino-2-cyanoacetate.

The condensation assistants may be used solely or as a mixture bycombining two or more of them. Preferred examples of the condensationassistant include an N-hydroxydicarboxylic imide compound (such as HONSuand the like), and an N-hydroxybenzotriazole or N-hydroxybenzotriazinecompound (such as HOBt and the like).

The dehydration condensing agent and the condensation assistant arepreferably used by suitably combining. Examples of the combination ofthe dehydration condensing agent and the condensation assistant includeDCC-HONSu (HOBt or HOOBt) and WSCI-HONSu (HOBt or HOOBt).

The amount of the dehydration condensing agent used is generally in arange of from 0.7 to 5 mol, preferably from 0.8 to 2.5 mol, and furtherpreferably from 0.9 to 2.3 mol (e.g., 1 to 2 mol), per 1 mol in total ofthe peptide fragments in the case where a nonaqueous solvent containingno water is used. In a solvent containing water (aqueous solvent), inconsideration of deactivation of the dehydration condensing agent withwater, the amount of the dehydration condensing agent used is generallyin a range of from 2 to 500 mol, preferably from 5 to 250 mol, andfurther preferably from 10 to 125 mol, per 1 mol in total of the peptidefragments.

The amount of the condensation assistant used is generally in a range offrom 0.5 to 5 mol, preferably from 0.7 to 2 mol, and further preferablyfrom 0.8 to 1.5 mol, irrespective of the kind of the solvent.

In the condensation reaction, the pH of the reaction system may becontrolled, and a base that does not participate the reaction may beadded. The pH can be controlled generally with an inorganic base (suchas sodium hydroxide, potassium hydroxide, sodium carbonate, sodiumhydrogencarbonate and the like), an organic base, an inorganic acid(such as hydrochloric acid and the like), or an organic acid, and the pHof the reaction solution is generally controlled around neutral (pH ofabout from 6 to 8). Examples of the base that does not participate thecondensation reaction include a tertiary amine compound, for example, atrialkylamine compound, such as trimethylamine, triethylamine,diisopropylethylamine and the like, a heterocyclic tertiary aminecompound, such as N-methylmorpholine, pyridine and the like, and thelike. The amount of the base used is generally selected from a range offrom 1 to 2 times the total molar number of the peptide fragments.

In the polypeptide thus obtained, the reagents used in the reactionremain. These affect the platelet aggregation inducing activity of theactive ingredient polypeptide and the function of platelets themselves,and therefore the reagents remaining are preferably removed by a knownmethod, such as a dialysis method, a column method, an ultrafiltrationmethod and the like. In consideration of stability and handling facilityof the polypeptide, it is preferred that the reaction solvent isreplaced with a preservation solvent. The reaction solvent can bereplaced with the target preservation solvent by using the targetpreservation solvent as a dialysis external fluid in the dialysismethod, or by using the target preservation solvent as a mobile phase inthe column method.

The preservation solvent is not particularly limited as far as it cansuppress alterations of the physical properties and biologicalproperties of the resulting active ingredient polypeptide. Examplesthereof include water, physiological saline and a buffer having abuffering function in a range of from weak acid to weak alkali. It ispreferred that such a substance is not contained that affects thefunction of platelets themselves and the blood coagulation factors.Examples of the substance that affects the function of plateletsthemselves include a chelating agent that chelates calcium ions in theblood plasma, such as ethylenediaminetetraacetic acid, sodium citrateand the like, an antiplatelet agent that depresses the function ofplatelets, and a substance that activates platelets. Examples of thesubstance that affects the blood coagulation factors include a substancecontaining calcium ions, which accelerate coagulation of blood orplatelet-rich plasma having been subjected to anticoagulation treatment.

The active ingredient polypeptide has platelet aggregation inducingactivity. Accordingly, the platelet aggregation inducing substance ofthe invention containing the same can be used for evaluation ofaggregation capacity of platelets. The method for measuring aggregationcapacity of platelets is not particularly limited as far as it is amethod using an inducing substance. Examples of the usable methodsinclude an absorbance method (transmitted light method) where the extentof aggregation of platelets is determined by absorbance (transmittance),an impedance method where it is determined by change of electricresistance between platinum electrodes, a particle counting method wherethe extent of aggregation of platelets is estimated from the number ofsingle platelets that do not participate aggregation of platelets byusing a particle measuring equipment, a light scattering method usingscattered light, a micromesh filter method using a filter, and the like.

The transmitted light method is being commonly employed clinically. Thetransmitted light method employs the following principle. Platelet-richplasma (which may be hereinafter referred to as “PRP”) is obtained bycentrifugation, and then platelet-poor plasma (which may be hereinafterreferred to as “PPP”) is obtained by centrifugation. The number ofplatelets in the PRP is controlled to from 200,000 to 400,000 μL⁻¹, anda prescribed amount (from 200 to 500 μL) of the PRP and the PPP areplaced in a cuvet and heated to 37° C., to which the inducing substanceis added under stirring with a magnetic stirrer. Thereafter, thetransmittance of the PRP increased associated with aggregation ismeasured by a spectrophotometer with the PPP as a control. As evaluationof aggregation of platelets, the maximum value of the change rate of thetransmittance, the maximum change rate of the platelet aggregationcurve, and the like are used, and such a method may be used that usestwo or three kinds of concentrations from low concentration to highconcentration. In general, a test using two concentrations is beingfrequently employed, and commercially available collagen has a lowconcentration of from 0.2 to 1 μg/mL (final concentration) or a highconcentration of from 2 to 4 μg/mL (final concentration).

In the case where the platelet aggregation inducing substance of theinvention is applied to the transmitted light method, the aforementionedoperation may be used except for adjustment of the concentration. In thecase where the concentration of the platelet aggregation inducingsubstance of the invention is adjusted, a diluent may be arbitrarilyselected from water, physiological saline and a buffer having the samecomposition as the preservation fluid, and is preferably water orphysiological saline. A solution diluted with water at a finalconcentration of 0.05 μg/mL has an inducing capability of aggregation ofplatelets that is equivalent to commercially available collagen(produced by MC Medical, Inc.) at 2 μg/mL.

The platelet aggregation inducing substance of the invention hassufficiently high platelet aggregation inducing activity as compared tocommercially available collagen, and has no necessity of crosslinking.The active ingredient polypeptide used in the invention isnon-crosslinked, and thus there is no influence on the function ofplatelets themselves due to a remaining crosslinking agent, whereby theaggregation capacity of platelets can be precisely evaluated.Furthermore, the process can be largely reduced owing to the absence ofcrosslinking.

The platelet aggregation inducing substance of the invention hasadhesion capability to platelets and thus can be used for evaluation ofadhesion capability of platelets, a verification test of adhesion ofplatelets to collagen under flowing, and the like. As a method formeasuring the aggregation capacity of platelets, a Baumgartner method, amethod using a glass bead tube with negative charge, and the like havebeen known, and the method is not limited as far as the method does notuse an intravascular subcutaneous tissue, examples of which include acollagen-coated glass bead method with glass beads or the like coatedwith collagen, and the like. Examples of the method for verifyingadhesion of platelets to collagen under flowing include a flow chambermethod using a chamber of glass or the like coated with collagen inside,the use thereof by coating on an inner surface of a capillary tube orthe like, and the like. A method for immobilizing collagen to beads orthe like may be arbitrarily selected from a method for immobilizing withcovalent bond and a method for immobilizing without covalent bond.

Example

The invention will be described in more detail with reference toexamples below. However, the invention is not limited to the examples.

Experimental Example 1 Synthesis of Polypeptide

1 g of a peptide fragment represented by Pro-Hyp-Gly synthesized by aliquid phase method was dissolved in 20 mL of a 10 mM phosphate buffer(pH 7.4), and cooled to 4° C. 473 mg of 1-hydroxybenzotriazole and 3.35g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride at 4°C. were added thereto, followed by stirring at 4° C. for 2 hours andthen stirring for 46 hours after heating to 20° C. After completing thereaction, the reaction solution was diluted twice with extra-pure water,and the resulting solution was placed in a cellulose tube for dialysis(UC27-32-100, produced by Viscase Corporation) and was dialyzed against2 L of pure water for 48 hours. The pure water was exchanged four timeswith an interval of 6 hours or more.

After dialyzing, the solution was diluted 50 times (volume ratio) withwater and subjected to gel permeation chromatography (high performanceliquid chromatography: 1100 Series produced by Agilent Technologies,column: OHpak SB-806m HQ produced by Shodex, flow rate: 1.0 mL/min,mobile phase: 20 mM potassium phosphate buffer (pH 3.0)/methanol=8/2(volume ratio)), and thus a peak of a polypeptide was observed in arange of molecular weight of from 10,000 to 500,000,000.

Separately, the solution after dialyzing was diluted 60 times (volumeratio) with water and measured for circular dichroic spectrum (circulardichroic polarimeter J-820, produced by Jasco Corporation, light pathlength: 1 mm), whereby positive Cotton effect was observed at 225 nm,and negative Cotton effect was observed at 198 nm.

Experimental Example 2

4.5 mL of blood was collected from a subject A with a vacuum bloodcollection tube containing 0.5 mL of 3.8 wt % by weight sodium citrate,and was well mixed to attain anticoagulation treatment. Subsequently,the blood having been subjected to anticoagulation treatment wassubjected to centrifugation at 100 g for 15 minutes, and a supernatantwas collected as PRP. Centrifugation was further performed at 2,000 gfor 15 minutes, and a supernatant was collected as PPP. The aggregationcapacity was evaluated in such a manner that the time course of thetransmittance after adding the polypeptide solution prepared inExperimental Example 1 was recorded by using a platelet aggregationmeasuring apparatus, Easy TRACER ET-800 (produced by Tokyo Koden Co.,Ltd.), with the transmittance of the PRP before adding an aggregationinducing substance being designated as 0% and the transmittance of thePPP being designated as 100%. The polypeptide solution was added to makea final concentration of the polypeptide of 2 μg/mL, and the time courseof the transmittance was recorded. A commercially available aggregationinducing substance of collagen (MC Medical, Inc.) as a control was addedto make a final concentration of collagen of 2 μg/mL, and the timecourse of the transmittance was recorded. The results are shown in FIG.1.

Experimental Example 3

The aggregation capacity of platelets was evaluated according to themethod disclosed in Experimental Example 2 except that 4.5 mL of bloodcollected from a subject B was used. The results are shown in FIG. 2.

Experimental Example 4

The aggregation capacity of platelets was evaluated according to themethod disclosed in Experimental Example 2 except that 4.5 mL of bloodcollected from a subject C was used, the final concentration of thepolypeptide was 2.0 μg/mL or 0.25 μg/mL, and the final concentration ofcollagen for comparison was 2.0 μg/mL or 0.25 μg/mL. The results areshown in FIGS. 3 and 4.

Experimental Example 5

The aggregation capacity of platelets was evaluated according to themethod disclosed in Experimental Example 2 except that 4.5 mL of bloodcollected from a subject D was used, the final concentration of thepolypeptide was 2.0 μg/mL, 0.25 μg/mL, 0.05 μg/mL or 0.02 μg/mL, and thefinal concentration of collagen for comparison was 2.0 μg/mL or 0.25μg/mL. The results are shown in FIGS. 5 and 6.

Experimental Example 6

The aggregation capacity of platelets was evaluated according to themethod disclosed in Experimental Example 2 except that 13.5 mL of bloodcollected from a subject A was used, the final concentration of thepolypeptide was 0.05 μg/mL, 0.025 μg/mL or 0.015 μg/mL, and the finalconcentration of collagen for comparison was 2.0 μg/mL or 0.25 μg/mL.The results are shown in FIGS. 7 and 8.

Experimental Example 7 Measurement of Aggregation of Platelets in thePresence or Absence of Aspirin

(1) Synthesis of Polypeptide

1 g of a peptide fragment represented by Pro-Hyp-Gly synthesized by aliquid phase method was dissolved in 20 mL of a 10 mM phosphate buffer(pH 7.4), and cooled to 4° C. 473 mg of 1-hydroxybenzotriazole and 3.35g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride at 4°C. were added thereto, followed by stirring at 4° C. for 2 hours andthen stirring for 46 hours after heating to 20° C. After completing thereaction, the reaction solution was placed in a cellulose tube fordialysis (UC27-32-100, produced by Viscase Corporation) and was dialyzedagainst 2 L of pure water for 48 hours. The pure water was exchangedfour times with an interval of 6 hours or more.

After dialyzing, the solution was diluted 100 times (volume ratio) withphysiological saline. The 100-time diluted solution was filtered (0.2μm, cellulose acetate membrane, 25CS020AS, available from Advantec ToyoCo., Ltd.) and then further diluted 50 times (volume ratio) withphysiological saline. After diluting 50 times, the concentration of thepolypeptide in the solution measured by an absorbance method(wavelength: 215 nm) was 0.29 μg/mL.

(2) Measurement of Aggregation of Platelets

PRP that was treated with aspirin (aspirin-treated PRP) and PRP that wasnot treated with aspirin (aspirin-non-treated PRP) were used formeasuring aggregation of platelets. PRP used was PRP obtained inExperimental Example 2, to which 1/10 amount (volume ratio) of a 10 mMaspirin solution, which was obtained by diluting aspirin(acetylsalicylic acid, produced by Wako Pure Chemical Industries, Ltd.,special grade reagent) with physiological saline, was added, followed byheating to 37° C. for 30 minutes, to provide the aspirin-treated PRP.1/10 amount (volume ratio) of physiological saline was added to PRPobtained in Experimental Example 2, followed by heating to 37° C. for 30minutes, to provide the aspirin-non-treated PRP.

The aggregation capacity was evaluated in such a manner that the timecourse of the transmittance after adding the polypeptide solutionprepared in this Experimental Example was recorded by using a plateletaggregation measuring apparatus, Easy TRACER ET-800 (produced by TokyoKoden Co., Ltd.), with the transmittance of the PRP before adding anaggregation inducing substance being designated as 0% and thetransmittance of the PPP being designated as 100%.

22 μL of the polypeptide solution (polypeptide concentration: 0.29μg/mL) was added to each of 200 μL of the aspirin-treated PRP and 200 μLof the aspirin-non-treated PRP, and the time course of the transmittancewas recorded. Subsequently, natural collagen, Collagen Reagent “Horm” (1mg/mL), available from Moriya Sangyo Co., Ltd., was diluted 10 times(volume ratio) with the accompanying diluent and was further diluted 5times (volume ratio) with physiological saline to provide a solution(collagen concentration: 20 μg/mL), which was added to each of 200 μL ofthe aspirin-treated PRP and 200 μL of the aspirin-non-treated PRP, andthe time course of the transmittance was recorded. The results are shownin FIG. 9.

In the case where natural collagen was used as an aggregation inducingsubstance, no aggregation of platelets was observed for theaspirin-treated PRP although aggregation of platelets was found for theaspirin-non-treated PRP. In the case where the polypeptide of thisExperimental Example was used, on the other hand, aggregation ofplatelets was observed for both the aspirin-treated PRP and theaspirin-non-treated PRP even with the concentration thereof of about1/70 of the natural collagen.

INDUSTRIAL APPLICABILITY

The thrombin variant of the invention is significantly lowered inaffinity to a heparin-like substance (heparan sulfate), thrombomodulinand/or integrin, which are present on the blood vessel wall, and hashigh antithrombotic capacity, and thus it can be effectively used asremedy for thrombotic disease and the like without side effect.

The invention claimed is:
 1. A method of inducing platelet aggregation,comprising adding, in a solution form, to blood or platelet-rich plasma,an un-crosslinked polypeptide having a peptide fragment (component A)represented by formula (1):-(Pro-X-Gly)_(n)-  (1) where n represents an integer of from 20 to5,000, and wherein the peptide of formula (1) is selected from the groupconsisting of: (Pro-Pro-Gly)- (Component B) and -(Pro-Hyp-Gly)-(Component C), wherein the molar ratio of the Component B:Component C insaid peptide fragment is 10/90 to 0/100, and wherein said un-crosslinkedpolypeptide is capable of inducing said platelet aggregation even in thepresence of an inhibitory amount of aspirin, as compared to collagen. 2.The method according to claim 1, wherein the polypeptide furthercomprises, as a component D, at least one selected from the groupconsisting of a peptide unit represented by formula (4), a peptide unitrepresented by formula (5) and a peptide fragment containing at leastone of the peptide units:—(Y₁—Y₂-Gly)-  (4)-(Pro-Z₁-Gly-Z₂-Ala-Gly)-(SEQ ID NO:13)  (5) wherein Y₁ represents Aspor Glu, which may have a carboxyl group at the γ-position; Y₂ representsPro or Hyp; Z₁ represents Gln, Asn, Leu, He, Val or Ala; Z₂ representsIle or Leu, and the peptide unit represented by formula (5) is selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
 3. The methodaccording to claim 2, wherein a molar ratio of the peptide fragment(component A) to the component D is in a range of from 99/1 to 30/70. 4.The method according to claim 1, wherein the polypeptide exhibitspositive Cotton effect at a wavelength of from 220 to 230 nm andnegative Cotton effect at a wavelength of from 195 to 205 nm in acircular dichroic spectrum.
 5. The method according to claim 1, whereinthe polypeptide has a molecular weight in a range of from 10,000 to500,000,000.
 6. The method according to claim 1, wherein the polypeptidehas a particle diameter in a range of from 0.01 to 1,000 μm.
 7. Themethod according to claim 1, wherein the polypeptide has a viscosity ina range of from 10 to 10,000 mPa·s, wherein the viscosity is measuredfor a 1 wt % aqueous solution of the polypeptide under a condition of20° C. with an E-type viscometer.
 8. The method according to claim 1,wherein the polypeptide is added in presence of aspirin.